Fibrinolytic enzyme from agrocybe aegerita and a preparation method thereof

Abstract

A fibrinolytic enzyme from Agrocybe aegerita and a preparation method thereof. The fibrinolytic enzyme from Agrocybe aegerita is composed of two subunits A and B, the molecular weight of the subunit A is 31.4 kDa; the molecular weight of the subunit B is 21.2 kDa; the twelve amino acids sequence of the N-terminal of the subunit A is shown in SEQ ID No. 1; the twelve amino acids sequence of the N-terminal of the subunit B is shown in SEQ ID No. 2. The optimal temperature for the fibrinolytic enzyme from Agrocybe aegerita provided in the invention is 47° C., which can maintain good activity at physiological pH of human. Fe2+ at different concentrations shows obvious inhibiting effects on the fibrinolytic enzyme from Agrocybe aegerita. The fibrinolytic enzyme from Agrocybe aegerita exhibits good thrombolytic properties, thus providing a research basis for the preparation of thrombolytic drugs and/or functional foods.

1 . A fibrinolytic enzyme from  Agrocybe aegerita , wherein the fibrinolytic enzyme from  Agrocybe aegerita  is composed of two subunits A and B, the molecular weight of the subunit A is 31.4 kDa; the molecular weight of the subunit B is 21.2 kDa; the twelve amino acids sequence of the N-terminal of the subunit A is shown in SEQ ID No. 1; the twelve amino acids sequence of the N-terminal of the subunit B is shown in SEQ ID No. 2. 
     
     
         2 . A preparation method for the fibrinolytic enzyme from  Agrocybe aegerita  of  claim 1 , comprising: extracting  Agrocybe aegerita  fruiting bodies with normal saline, to obtain an extract; performing a liquid-solid separation on the extract, the fibrinolytic enzyme in the resulting crude enzyme solution is separated successively through salting precipitation, gel chromatography, weak cation exchange chromatography, hydrophobic interaction chromatography and strong cation exchange chromatography. 
     
     
         3 . The preparation method according to  claim 2 , wherein the ratio between the mass of  Agrocybe aegerita  fruiting bodies and the volume of normal saline is 1 kg:(8 to 12) L. 
     
     
         4 . The preparation method according to  claim 2 , wherein the conditions for extraction comprise: the extraction time is 5 to 7 hours, and the extraction temperature is 3 to 5° C. 
     
     
         5 . The preparation method according to  claim 2 , wherein the conditions for separation over the gel chromatographic column comprise: the eluent is a PBS buffer solution at 0.01 to 0.03 mol/L, the flow rate is 4 to 6 mL/min; and the pH of the PBS buffer is independently 6.0. 
     
     
         6 . The preparation method according to  claim 2 , wherein the separation by weak cation exchange chromatography is performed by linear elution, the starting buffer is the PBS buffer at 0.01 to 0.03 mol/L, the eluent is the PBS buffer at 0.01 to 0.03 mol/L containing 04 to 1 mol/L of NaCl; the pH of the PBS buffer is independently 6.0; and the flow rate is independently 1 to 3 mL/min. 
     
     
         7 . The preparation method according to  claim 2 , wherein the separation by hydrophobic interaction chromatography is performed by linear elution, the starting buffer is the PBS buffer at 0.01 to 0.03 mol/L containing ammonium sulfate at a saturation of 10%, the eluent is the PBS buffer at 0.01 to 0.03 mol/L containing ammonium sulfate at a saturation of 10% to 0%; and the pH of the PBS buffer is independently 7.4. 
     
     
         8 . The preparation method according to  claim 2 , wherein the separation by strong cation exchange chromatography is performed by linear elution, the starting buffer is the PBS buffer at 0.01 to 0.03 mol/L, the eluent is the PBS buffer at 0.01 to 0.03 mol/L containing 04 to 1 mol/L of NaCl; and the pH of the PBS buffer Is independently 5.5.